Erythritol disrupts neurodevelopmental signaling and behavior in zebrafish via shh/nodal and notch pathways

The safety of erythritol, a widely used zero-calorie sugar substitute, concerning neurodevelopment is yet to be fully understood. In this study, we utilized EmbryoNet, a deep learning-driven embryonic phenotyping platform, to assess the neurodevelopmental toxicity of erythritol at concentrations of 3, 5, and 7 mg/mL, which adhere to FDA standards. Our findings demonstrated specific disruptions in the shh and nodal signaling pathways, critical regulators of vertebrate neural patterning. Experimental validation revealed that erythritol exposure led to microcephaly, reduced neural stem cells, and increased brain cell apoptosis in larvae. Mechanistically, reduced ddx3x gene expression in zebrafish inhibited the expression of Notch ligands (dll4, jag1a) and receptors (notch1b, notch2) by disrupting their interactions with crebbp, thereby triggering a range of effects. These include neurotransmitter disorders such as a GABA/glutamate imbalance (up-regulation of gad1b and down-regulation of gabra2a expression) and dopamine metabolic abnormalities (up-regulation of dat and down-regulation of comt gene expression). Behavioral analyses further corroborated these findings, showing erythritol-induced abnormalities including heightened thigmotaxis, stereotyped repetitive behaviors, and increased locomotor activity. These findings highlight the potential of erythritol to induce neurodevelopmental impairments through multifaceted interactions, implicating both autism-related genes (nomo1, shank3a, shank3b, tanc2a, tanc2b) and ADHD-related genes (lphn3.1, lphn3.2, per1b). This study underscores the harmful neurodevelopmental consequences of erythritol and establishes a framework for its rational and safe utilization.